Ca2+ elevations disrupt interactions between intraflagellar transport and the flagella membrane in Chlamydomonas

ABSTRACTThe movement of ciliary membrane proteins is directed by transient interactions with intraflagellar transport (IFT) trains. The green alga Chlamydomonas has adapted this process for gliding motility, using retrograde IFT motors to move adhesive glycoproteins in the flagella membrane. Ca2+ signalling contributes directly to the gliding process, although uncertainty remains over the mechanism through which it acts. Here, we show that flagella Ca2+ elevations initiate the movement of paused retrograde IFT trains, which accumulate at the distal end of adherent flagella, but do not influence other IFT processes. On highly adherent surfaces, flagella exhibit high-frequency Ca2+ elevations that prevent the accumulation of paused retrograde IFT trains. Flagella Ca2+ elevations disrupt the IFT-dependent movement of microspheres along the flagella membrane, suggesting that Ca2+ acts by directly disrupting an interaction between retrograde IFT trains and flagella membrane glycoproteins. By regulating the extent to which glycoproteins on the flagella surface interact with IFT motor proteins on the axoneme, this signalling mechanism allows precise control of traction force and gliding motility in adherent flagella.

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Flagella Ca2+ elevations regulate pausing of retrograde intraflagellar transport trains in adherent Chlamydomonas flagella

10.1101/2020.08.17.240366 ◽

2020 ◽

Author(s):

Cecile Fort ◽

Peter Collingridge ◽

Colin Brownlee ◽

Glen Wheeler

Keyword(s):

Membrane Proteins ◽

Green Alga ◽

High Frequency ◽

Intraflagellar Transport ◽

Gliding Motility ◽

Membrane Glycoproteins ◽

Ciliary Membrane ◽

Process Uncertainty ◽

Temporal Properties ◽

Transient Interactions

AbstractThe movement of ciliary membrane proteins is directed by transient interactions with intraflagellar transport (IFT) trains. The green alga Chlamydomonas has adapted this process for gliding motility, using IFT to move adhesive glycoproteins (FMG-1B) in the flagella membrane. Although Ca2+ signalling contributes directly to the gliding process, uncertainty remains over the mechanisms through which Ca2+ acts to influence the movement of IFT trains. Here we show that flagella Ca2+ elevations regulate IFT primarily by initiating the movement of paused retrograde IFT trains. Flagella Ca2+ elevations exhibit complex spatial and temporal properties, including high frequency repetitive Ca2+ elevations that prevent the accumulation of paused retrograde IFT trains. We show that flagella Ca2+ elevations disrupt the IFT-dependent movement of microspheres along the flagella membrane. The results suggest that flagella Ca2+ elevations directly disrupt the interaction between retrograde IFT particles and flagella membrane glycoproteins to modulate gliding motility and the adhesion of the flagellum to a surface.

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A WDR35-dependent coat protein complex transports ciliary membrane cargo vesicles to cilia

eLife ◽

10.7554/elife.69786 ◽

2021 ◽

Vol 10 ◽

Author(s):

Tooba Quidwai ◽

Jiaolong Wang ◽

Emma A Hall ◽

Narcis A Petriman ◽

Weihua Leng ◽

...

Keyword(s):

Coat Protein ◽

Membrane Proteins ◽

Sequence Homology ◽

Protein Complex ◽

Intraflagellar Transport ◽

Retrograde Transport ◽

Ciliary Membrane ◽

Mouse Mutants ◽

Core Components

Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the non-core IFT-A components are degraded and core components accumulate at the ciliary base. We reveal deep sequence homology of WDR35 and other IFT-A subunits to α and ß' COPI coatomer subunits, and demonstrate an accumulation of 'coat-less' vesicles which fail to fuse with Wdr35 mutant cilia. We determine that recombinant non-core IFT-As can bind directly to lipids and provide the first in-situ evidence of a novel coat function for WDR35, likely with other IFT-A proteins, in delivering ciliary membrane cargo necessary for cilia elongation.

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Intraflagellar transport drives flagellar surface motility

eLife ◽

10.7554/elife.00744 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 54

Author(s):

Sheng Min Shih ◽

Benjamin D Engel ◽

Fatih Kocabas ◽

Thomas Bilyard ◽

Arne Gennerich ◽

...

Keyword(s):

Intraflagellar Transport ◽

Gliding Motility ◽

Solid Surfaces ◽

Dependent Manner ◽

Surface Adhesion ◽

Membrane Glycoproteins ◽

Flagellar Membrane ◽

Surface Motility ◽

Cilia And Flagella ◽

Ciliary Signaling

The assembly and maintenance of all cilia and flagella require intraflagellar transport (IFT) along the axoneme. IFT has been implicated in sensory and motile ciliary functions, but the mechanisms of this relationship remain unclear. Here, we used Chlamydomonas flagellar surface motility (FSM) as a model to test whether IFT provides force for gliding of cells across solid surfaces. We show that IFT trains are coupled to flagellar membrane glycoproteins (FMGs) in a Ca2+-dependent manner. IFT trains transiently pause through surface adhesion of their FMG cargos, and dynein-1b motors pull the cell towards the distal tip of the axoneme. Each train is transported by at least four motors, with only one type of motor active at a time. Our results demonstrate the mechanism of Chlamydomonas gliding motility and suggest that IFT plays a major role in adhesion-induced ciliary signaling pathways.

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Single molecule imaging reveals a major role for diffusion in the exploration of ciliary space by signaling receptors

eLife ◽

10.7554/elife.00654 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 86

Author(s):

Fan Ye ◽

David K Breslow ◽

Elena F Koslover ◽

Andrew J Spakowitz ◽

W James Nelson ◽

...

Keyword(s):

Membrane Proteins ◽

Active Transport ◽

Single Molecule ◽

Primary Cilia ◽

Somatostatin Receptor ◽

Intraflagellar Transport ◽

Signal Propagation ◽

Protein Diffusion ◽

Ciliary Membrane ◽

Signaling Receptors

The dynamic organization of signaling cascades inside primary cilia is key to signal propagation. Yet little is known about the dynamics of ciliary membrane proteins besides a possible role for motor-driven Intraflagellar Transport (IFT). To characterize these dynamics, we imaged single molecules of Somatostatin Receptor 3 (SSTR3, a GPCR) and Smoothened (Smo, a Hedgehog signal transducer) in the ciliary membrane. While IFT trains moved processively from one end of the cilium to the other, single SSTR3 and Smo underwent mostly diffusive behavior interspersed with short periods of directional movements. Statistical subtraction of instant velocities revealed that SSTR3 and Smo spent less than a third of their time undergoing active transport. Finally, SSTR3 and IFT movements could be uncoupled by perturbing either membrane protein diffusion or active transport. Thus ciliary membrane proteins move predominantly by diffusion, and attachment to IFT trains is transient and stochastic rather than processive or spatially determined.

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A WDR35-dependent coatomer transports ciliary membrane proteins from the Golgi to the cilia

10.1101/2020.12.22.423978 ◽

2020 ◽

Author(s):

Tooba Quidwai ◽

Emma A. Hall ◽

Margaret A. Keighren ◽

Weihua Leng ◽

Petra Kiesel ◽

...

Keyword(s):

Membrane Proteins ◽

Diffusion Barrier ◽

Sequence Homology ◽

Intraflagellar Transport ◽

Structural Similarity ◽

Retrograde Transport ◽

Ciliary Membrane ◽

Mouse Mutants ◽

Core Components

AbstractIntraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia and across the diffusion barrier is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the IFT-A peripheral components are degraded and core components accumulate at the transition zone. We reveal deep sequence homology and structural similarity of WDR35 and other IFT-As to the coatomer COPI proteins α and β′, and demonstrate an accumulation of ‘coat-less’ vesicles which fail to fuse with Wdr35 mutant cilia. Our data provides the first in situ evidence of a novel coatomer function for WDR35 likely with other IFT-A proteins in delivering ciliary membrane cargo from the Golgi necessary for cilia elongation.

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Trafficking of ciliary membrane proteins by the intraflagellar transport/BBSome machinery

Essays in Biochemistry ◽

10.1042/ebc20180030 ◽

2018 ◽

Vol 62 (6) ◽

pp. 753-763 ◽

Cited By ~ 39

Author(s):

Jenna L. Wingfield ◽

Karl-Ferdinand Lechtreck ◽

Esben Lorentzen

Keyword(s):

Membrane Proteins ◽

Molecular Motors ◽

Intraflagellar Transport ◽

Inherited Disease ◽

Cell Organelles ◽

Bardet Biedl Syndrome ◽

Ciliary Membrane ◽

Peripheral Membrane Proteins ◽

Abnormal Accumulation ◽

And Function

Bardet–Biedl syndrome (BBS) is a rare inherited disease caused by defects in the BBSome, an octameric complex of BBS proteins. The BBSome is conserved in most organisms with cilia, which are microtubule (MT)-based cell organelles that protrude from the cell surface and function in motility and sensing. Cilia assembly, maintenance, and function require intraflagellar transport (IFT), a bidirectional motility of multi-megadalton IFT trains propelled by molecular motors along the ciliary MTs. IFT has been shown to transport structural proteins, including tubulin, into growing cilia. The BBSome is an adapter for the transport of ciliary membrane proteins and cycles through cilia via IFT. While both the loss and the abnormal accumulation of ciliary membrane proteins have been observed in bbs mutants, recent data converge on a model where the BBSome mainly functions as a cargo adapter for the removal of certain transmembrane and peripheral membrane proteins from cilia. Here, we review recent data on the ultrastructure of the BBSome and how the BBSome recognizes its cargoes and mediates their removal from cilia.

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A Novel Reconstitution Method for Inducing the Formation of Regular 2-Dimensional Arrays of Membrane Proteins and Lipids

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102845 ◽

1988 ◽

Vol 46 ◽

pp. 152-153 ◽

Cited By ~ 1

Author(s):

A. Engel ◽

A. Holzenburg ◽

K. Stauffer ◽

J. Rosenbusch ◽

U. Aebi

Keyword(s):

Membrane Proteins ◽

Flow Rate ◽

Lipid Membranes ◽

Functional Characterization ◽

Dimensional Structure ◽

Electron Crystallography ◽

Peltier Element ◽

Protein Ratio ◽

Precise Control ◽

3 Dimensional

Reconstitution of solubilized and purified membrane proteins in the presence of phospholipids into vesicles allows their functions to be studied by simple bulk measurements (e.g. diffusion of differently sized solutes) or by conductance measurements after transformation into planar membranes. On the other hand, reconstitution into regular protein-lipid arrays, usually forming at a specific lipid-to-protein ratio, provides the basis for determining the 3-dimensional structure of membrane proteins employing the tools of electron crystallography.To refine reconstitution conditions for reproducibly inducing formation of large and highly ordered protein-lipid membranes that are suitable for both electron crystallography and patch clamping experiments aimed at their functional characterization, we built a flow-dialysis device that allows precise control of temperature and flow-rate (Fig. 1). The flow rate is generated by a peristaltic pump and can be adjusted from 1 to 500 ml/h. The dialysis buffer is brought to a preselected temperature during its travel through a meandering path before it enters the dialysis reservoir. A Z-80 based computer controls a Peltier element allowing the temperature profile to be programmed as function of time.

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Ciliary membrane proteins traffic through the Golgi via a Rabep1/GGA1/Arl3-dependent mechanism

Nature Communications ◽

10.1038/ncomms6482 ◽

2014 ◽

Vol 5 (1) ◽

Cited By ~ 72

Author(s):

Hyunho Kim ◽

Hangxue Xu ◽

Qin Yao ◽

Weizhe Li ◽

Qiong Huang ◽

...

Keyword(s):

Membrane Proteins ◽

Ciliary Membrane ◽

Dependent Mechanism

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Trafficking to the primary cilium membrane

Molecular Biology of the Cell ◽

10.1091/mbc.e16-07-0505 ◽

2017 ◽

Vol 28 (2) ◽

pp. 233-239 ◽

Cited By ~ 24

Author(s):

Saikat Mukhopadhyay ◽

Hemant B. Badgandi ◽

Sun-hee Hwang ◽

Bandarigoda Somatilaka ◽

Issei S. Shimada ◽

...

Keyword(s):

Membrane Proteins ◽

Signaling Pathways ◽

Primary Cilium ◽

Hedgehog Signaling ◽

Diffusion Barriers ◽

Disease Pathogenesis ◽

Ciliary Membrane ◽

Multiple Organs ◽

Sensory Reception

The primary cilium has been found to be associated with a number of cellular signaling pathways, such as vertebrate hedgehog signaling, and implicated in the pathogenesis of diseases affecting multiple organs, including the neural tube, kidney, and brain. The primary cilium is the site where a subset of the cell's membrane proteins is enriched. However, pathways that target and concentrate membrane proteins in cilia are not well understood. Processes determining the level of proteins in the ciliary membrane include entry into the compartment, removal, and retention by diffusion barriers such as the transition zone. Proteins that are concentrated in the ciliary membrane are also localized to other cellular sites. Thus it is critical to determine the particular role for ciliary compartmentalization in sensory reception and signaling pathways. Here we provide a brief overview of our current understanding of compartmentalization of proteins in the ciliary membrane and the dynamics of trafficking into and out of the cilium. We also discuss major unanswered questions regarding the role that defects in ciliary compartmentalization might play in disease pathogenesis. Understanding the trafficking mechanisms that underlie the role of ciliary compartmentalization in signaling might provide unique approaches for intervention in progressive ciliopathies.

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Further studies on the interaction between human platelet membrane glycoproteins IIb and IIIa in triton X-100

Blood ◽

10.1182/blood.v60.4.894.894 ◽

1982 ◽

Vol 60 (4) ◽

pp. 894-904 ◽

Cited By ~ 15

Author(s):

D Pidard ◽

JP Rosa ◽

TJ Kunicki ◽

AT Nurden

Keyword(s):

Membrane Proteins ◽

Divalent Cation ◽

Human Platelet ◽

Polyacrylamide Gel Electrophoresis ◽

Indirect Evidence ◽

Human Platelets ◽

Platelet Membrane ◽

Membrane Glycoproteins ◽

Nonionic Detergent ◽

Triton X

Abstract Analysis of human platelet membrane proteins by crossed immunoelectrophoresis (CIE) in the presence of Triton X-100 (TX-100) has previously shown that glycoproteins (GP) IIb and IIIa are located in a single immunoprecipitate, band 16.2 To investigate whether IIb and IIIa are associated in a complex, we have analyzed TX-100-solubilized 125I-labeled membrane proteins by density gradient ultracentrifugation using 10%-40% sucrose gradients containing the nonionic detergent. studies were performed using soluble proteins derived from membranes isolated in the presence or absence of EDTA. Analysis of gradient fractions by SDS-polyacrylamide gel electrophoresis showed that in the absence of divalent cation chelation, GP IIb and IIIa penetrated well into the gradient (fractions 15–17). Analysis of fractions 15–17 by CIE revealed the presence of band 16. In contrast, when the membrane proteins were incubated with EDTA prior to or after TX-100 solubilization, IIb and IIIa remained near the top of the gradient (fractions 8–11) and gave separate immunoprecipitates during CIE. Incubation of washed platelet lysates with leupeptin, an inhibitor of the Ca2+-dependent protease of human platelets, had no effect on the shape of the band 16 immunoprecipitate. Thus, for the first time, direct evidence has been obtained that GP IIb and IIIa may form a divalent cation-mediated complex. Calibration of the sedimentation profiles using proteins of known molecular weight suggests that the complex is of limited size. Indirect evidence suggests that the complex is a heterodimer.

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